The invention relates to reagents and methods for detecting and/or quantifying the phosphorylation state and/or level of proteins in samples.
The availability of complete genome sequences is moving biological research to an era where cellular systems are analyzed as a whole rather than analyzing the individual components. While genome sequences and global gene expression measurements at the mRNA level opens the door to important biological advances, much of the understanding of cellular systems and the roles of the constituents will arise from proteomics (Wasinger et al., Electrophoresis 16:1090-1094, 1995). Proteomics, the analysis of the entire complement of proteins expressed by a cell, tissue type, or organ, provides the most informative characterization of the cell because proteins are the primary players that carry out nearly all processes within the cell. A key aspect to successful proteomic measurements is the ability to precisely measure protein abundance changes in a high throughput manner so as to allow the effects of many xe2x80x9cperturbationsxe2x80x9d upon, or changes to, a cell type, tissue type or organ, to be determined in a rapid fashion (Pafterson, Curr. Opin. Biotechnol. 11:413-418, 2000). An inherent goal of proteomic studies is to provide a greater understanding of the function of proteins in a global, cellular context, along with the more conventionally delineated molecular function. A greater understanding at the level of cellular systems will provide, for example, a stronger basis for understanding complex biological pathways and the nature of diseases. The global understanding of cellular systems provided by proteomic investigations will provide numerous opportunities unlikely to originate from the present paradigm of xe2x80x9csinglexe2x80x9d protein characterization methodologies.
Though methods to allow global measurements of gene expression at the mRNA level have been developed (Schena et al., Science 270:467-470, 1995 and Khodursky et al., Proc. Natl. Acad. Sci. USA 97:12170-12175, 2000), such methods do not provide direct measurements of protein abundance (Gygi et al., Proc. Natl. Acad. Sci. USA 97:9390-9395, 2000). Clearly, only global analysis of gene expression at the protein level provides information about the roles of individual gene products and their involvement in cellular pathways. Information relating solely to the abundance of a particular protein within a cell fails to provide information relating to the xe2x80x9cprocessed statexe2x80x9d of the protein. The xe2x80x9cprocessed statexe2x80x9d refers to the level and/or type of post-translational modifications that are displayed by the functional protein. For example many proteins are initially translated in an inactive form and upon subsequent proteolysis, the addition of sugar moieties, phosphate groups, methyl groups, carboxyl groups, or other additional groups so they gain biological function. Information relating to the xe2x80x9cprocessed statexe2x80x9d of a given protein is necessary and, hence, methods of detecting the active state of proteins are important for furthering the understanding of intercellular signaling and for developing new and useful interventions and therapeutics.
The reversible phosphorylation of proteins plays a key role in transducing extracellular signals into the cell. Many proteins that participate in cell signaling pathways are phosphorylated via enzymes known as kinases and dephosphorylated via phosphatases. Phosphate groups are added to, for example, tyrosine, serine, threonine, histidine, and/or lysine amino acid residues depending on the specificity of the kinase acting upon the target protein. To date several disease states have been linked to the abnormal phosphorylation/dephosphorylation of specific proteins. For example, the polymerization of phosphorylated tau protein allows for the formation of paired helical filaments that are characteristic of Alzheimer""s disease, and the hyperphosphorylation of retinoblastoma protein (pRB) has been reported to progress various tumors (Vanmechelen et al. Neurosci. Lett. 285:49-52, 2000, and Nakayama et al. Leuk. Res. 24:299-305, 2000, respectively).
The ability to quickly screen for irregularties in the phosphorylation state of proteins will further the understanding of intra and inter cellular signaling and lead to the development of improved diagnostics for the detection of various disease states.
The present disclosure provides reagents and methods for characterizing the phosphorylation state and/or level of a protein. Proteins can be post-transcriptionally modified such that they contain phosphate groups at either some, or all serine, threonine, tyrosine, histidine, and/or lysine amino acid residues. In many cases the extent to which a protein is phosphorylated determines it bioactivity, i.e., its ability to effect cell functions such as differentiation, division, and metabolism. Hence, the present disclosure provides powerful methods for diagnosing various diseases and for furthering the understanding of proteinxe2x80x94protein interactions.
One aspect of the invention provides methods of comparing the phosphorylation state of one or more proteins in two or more samples. Moreover, the disclosed methods allow for the generation of phosphorylation profiles, which detect changes in the phosphorylation states of individual proteins within samples, rather than merely detecting overall increases in the phosphorylation level of all of the proteins within a sample. These methods involve providing a substantially chemically identical and differentially isotopically labeled protein reactive reagent for each sample wherein the protein reactive reagent satisfies the formula:
B-L-PhRG 
wherein B is a binding agent that selectively binds to a capture reagent (CR), L is a linker group having one or more atoms that are differentially labeled with one or more isotopes, and PhRG is a phosphate reactive group that selectively reacts with amino acid residues that were formerly phosphorylated. Each sample is then reacted with one of the protein reactive reagents to provide proteins bound to the protein reactive agent, whereby the bound proteins are differentially labeled with the isotopes. The bound proteins of the samples are captured using the capture reagent that selectively binds the binding agent and the captured bound protein is then subsequently released from the capture reagent by disrupting the interaction between the binding agent and the capture reagent. The released, bound protein is then detected.
Another aspect of the invention provides methods for screening for therapeutics that alter the phosphorylation state of one or more proteins. These methods involve contacting at least one test sample containing a protein with the therapeutic and providing at least one control sample also containing an amount of the protein. One or more phosphate groups from one or more of the amino acid residues in the proteins in the test sample and control sample are removed and the proteins in both the test sample and the control are tagged with isotopically distinguishable B-L-PhRG that are substantially chemically identical. The level of phosphorylation can then be detected in the at least one test sample and the at least one control sample and the ability of the therapeutic to alter the phosphorylation of the protein can be determined.
Another aspect of the invention provides methods of comparing the level of tyrosine, serine, and threonine phosphorylation states of one or more proteins from two or more samples. Generally, these methods are practiced by sequentially removing phosphate groups from either the tyrosine residues, or the serine and threonine residues (the phosphate groups can be removed from either the serine and threonine first or the tyrosine phosphate groups can be removed first). Regardless of which phosphate group is removed first, a differentially isotopically labeled B-L-PhRG is subsequently used to xe2x80x9ctagxe2x80x9d the protein at the site formerly occupied by the phosphate group. The term xe2x80x9cisotopically labeledxe2x80x9d means that the L in the B-L-PhRG contains from about 0 to about 400, from about 1 to 300, from about 1 to about 250, from about 1 to about 200, from about 1 to about 150, from about 1 to 100, or from about 1 to about 50, isotopically different atoms. For example, the L will contain from about 0 to about 50 deuterium atoms substituted for hydrogen atoms. Thus the term xe2x80x9cdifferentially isotopically labeledxe2x80x9d refers to the fact that when two or more proteins, or two or more samples, are compared, each will be tagged with a B-L-PhRG complex that is isotopically distinguishable from the B-L-PhRG used to label the other protein, or the other sample. Hence, in the example above the serine and threonine residues from the first sample could, for example, be tagged with a B-L-PhRG that contains HSCH2CH2CH2SH and a second sample could, for example, be tagged with a B-L-PhRG that contains HSCD2CD2CD2SH. In another embodiment, the tyrosine residues from the first sample could be tagged with a B-L-PhRG that contains HSCH2CH2SH and a second sample could be tagged with a B-L-PhRG that contains HSCD2CD2SH.
Additionally the B of the B-L-PhRG could be chosen such that it was captured by different capture reagents. For example, allowing the tagged tyrosine residues to be captured in a first column chromatography step and the serine and threonine residues to be captured by a second column chromatography step.
The methods described herein can be applied to peptides (fragments of proteins) that are generated via enzymatic or chemical processing. The methods described herein can be also followed by protein sequencing via tandem mass spectrometry, chromatographic separation, or other suitable methods now known or developed in the future for such sequencing. Additionally, standards of known amounts of differentially isotopically labeled proteins can be introduced such that they provide an internal standard that aids in protein and/or peptide quantification.
The methods of this invention can also be used to identify and/or detect a plurality of proteins in a single sample.
Moreover, the methods described herein can be used to compare relative amounts of one or more proteins in two or more samples by individually tagging one or more proteins from two or more samples with isotopically distinguishable B-L-PhRG complexes and then combining the tagged proteins into one sample. The combined sample is then captured, isolated and the differentially isotopically labeled proteins are detected and/or quantified.
The methods described herein are especially useful for quantifying the phosphorylation state of membrane proteins, proteins originating from different organelles, and proteins from different subcellular fractions. Similarly, the methods described herein can be used to determine the phosphorylation state of proteins that results from treatment of samples with different environmental or nutritional conditions, different chemical or physical stimuli or at different times. In some cases, the difference in the phosphorylation state of a protein may be the result of the protein""s phosphorylation state in different disease states.
The binding agent can also be used to bind to fluorescently labeled conjugates. For example, streptavidin labeled with a fluorescent chelate can be used to bind to a biotinylated sample. The sample can then be detected via UV absorbance. This method can be used to detect the phosphorylation state of a protein that has been immunoprecipitated prior to being tagged at its phosphorylation sites. Used in this way, the phosphorylation state of a specific protein can be compared with a control sample without the need for protein sequencing, quantification or the use of antibodies selective for the phosphorylated protein itself.
The comparison methods disclosed herein can be also used for characterizing changes in the phosphorylation state of proteins in samples that have been treated with compounds such as nucleic acid sequences, proteins, protein fragments, and chemicals. Further, the methods are useful for detecting different phosphorylation states of proteins in xe2x80x9cdiseasedxe2x80x9d versus xe2x80x9cnormalxe2x80x9d specimens. When used in this context, the methods provided herein can be used as diagnostic tools to identify subjects that are suffering from diseases caused by protein phosphorylation abnormalities.
Another aspect of the methods described herein provides reagents for mass spectrometric analysis of proteins. As set forth above, the general formula of the mass spectrometry reagents of the present invention is B-L-PhRG, where B is a binding agent that selectively binds to a capture reagent, L is a linker group that has at least one isotopically labeled isotope (i.e., 2H, 13C, 15N, 17O, 18O, or 34S) and contains at least one phosphorylation (phosphate) reactive group (PhRG) that selectively labels proteins at one or more residues that were formerly occupied by phosphate groups. Such mass spectrometry reagents preferably satisfy the formula:
Bxe2x80x94B1xe2x80x94X1xe2x80x94(CH2)nxe2x80x94[X2xe2x80x94(CH2)m]Xxe2x80x94X3xe2x80x94(CH2)pxe2x80x94X4xe2x80x94B2-PhRG 
where: B is a binding agent, PhRG is a protein reactive group, and The linker group should be xe2x80x9cB1xe2x80x94X1 . . . B2xe2x80x9d and not the entire molecule (that is not including xe2x80x9cBxe2x80x9d or xe2x80x9cPhRGxe2x80x9d.
Bxe2x80x94B1xe2x80x94X1xe2x80x94CH2)nxe2x80x94[X2xe2x80x94(CH2)m]Xxe2x80x94X3xe2x80x94(CH2)pxe2x80x94X4xe2x80x94B2-PhRG is a linker group wherein: X1, X2, X3 and X4, and the X2 and other X2, are all independently selected from O, S, NH, NR, NRRxe2x80x2+, CO, COO, COS, Sxe2x80x94S, SO, SO2, CO-NRxe2x80x2, CS-NRxe2x80x2, Si-O, aryl or diaryl groups or X1-X4 may be absent; B1 and B2, are optional groups independently selected from COO, CO, CO-NRxe2x80x2, CS-NRxe2x80x2, (CH2)q-CONRxe2x80x2, (CH2)q-CS-NRxe2x80x2, or (CH2)q; n, m, p, q and x are whole numbers that can take values from 0 to about 100, where the sum of n+xm+p+q is less than about 100; R is an alkyl, alkenyl, alkynyl, alkoxy or an aryl group that is optionally substituted with one or more alkyl, alkenyl, alkynyl, or alkoxy groups; and Rxe2x80x2 is a hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or an aryl group that is optionally substituted with one or more alkyl, alkenyl, alkynyl, or alkoxy groups, wherein one or more of the CH2 groups in the linker can be optionally substituted with alkyl, alkenyl, alkoxy groups, an aryl group that is optionally substituted with one or more alkyl, alkenyl, alkynyl, or alkoxy groups, an acidic group, a basic group or a group carrying a permanent positive or negative charge; wherein one or more single bonds linking non-adjacent CH2 groups in the linker can be replaced with a double or a triple bond and wherein one or more of the atoms in the linker can be substituted with a stable isotope.
These and other aspects of the invention will become evident upon reading the following detailed description.